Method of producing l-glutamic acid from racemic glutamic acid



United States Patent Ofice Patented Oct. 10, 1961 3 003,921 METHOD OF PRODI ICING L-GLUTAMIC'ACID FROM RACEMIC GLUTA MHI ACID Shukuo Kinoshita, Masao Tanaka, andYo Kata, Tokyo, Japan, assignors to Kyowa Hakko Kogyo Co., Ltd, Tokyo, Japan, a corporation of Japan No Drawing. Filed-Sept. 12, 1960, Ser. No. 55,198 Claims priority, application Japan Sept. 11, 1959 17- Claims. (Cl. 195-29) The present invention relates to a method for producing L-glutamic acid from racemic glutamic acid by use of the enzymatic action of a microorganism. More parti'cularly this'invention relates to a method in which dehydration of L-glutamic acid (by the enzymatic action of Pseudomonas crucivz'ac) and racemization of remaining D-glutamic acid (by an aromatic or. heterocyclic aldehyde having in a position ortho to the aldehyde group a radial coordinatable with metal, and a metallic ion) are allowed to take place concurrently, thereby convertingracemic glutamic acid into L-Z-pyTroIidone-Srcarboxylic acid; the latter is then hydrolyzed to yield L glutarnic acid. L-glutamic acid produced is useful as a flavoring; agent or flavor enhancer in a variety of food products.

It is an object of the. present invention to obtain L- glutamic acid of a high purity with ease and in a higher yield from racemic glutamic acid by a short treatment, withoutconducting troublesome procedures, such as an optical resolution, etc. "Other objects and advantages areapparent from the ensuing description.

The. production of L-glutamic acid from racemic glutamic acid has heretofore been eliected. by resolving racemic glutamic acid into L- and D-isomers or their derivatives in a physical or biochemical way, racemizing the. D-glutamic acid remaining after the resolution, resolvingagain the racemized glutamic acid. in the same way, and repeating these procedures to convertthe whole racemic acid into theL-acid. However, such repeated.

procedures are not only very complex and time consuming, but also result in low yields of L-glutamic acid, this giving rise to various technical and economic difficulties.

In a copending application Serial No. 850,755, entitled Method for Producing L-Glutamic Acid From Racemic Glutamic Acid by Use of a: Micro-Organism, by S. K inoshita et' al;, filed onNovember' -i', 1959, there isproposed an'exceptionally superior method of resolving glutamic acid by. dehydrating L-glutamic acid with Pseudomonas cruciviae. Research" on the dehydration of L- glutamic acidand racemization of L-2.-pyrrolidone-5- carboxylict acid; resulting from said'd'ehydration, in the presenceof 1 an aromatic or heterocyclic aldehydehaving in itsortho-position a radical co-ordinatable withmetal and (2') a metallic ion havenow resulted inza novel methof. readily converting racemic. glutamicacid toL-glutamic'acid;

Research. on. the racemization of amino acid in the' presence of (1) an aromatic or heterocyclic aldehyde having in; its. ortho position: a: radical co-ordinatable with metal. and? (2) a metallic ion: was conducted: by Snellet alz, andthe reaction mechanism of theracemizationhas been clarified [J.. Amer. Chem. Soc. '76, 648' (1954)]. The explanation of. the mechanism. for? the. racemization:

of an amino acid involves the: formation: of a complex; compoundfrom the metallic ion and the aromatic or heterocyclic aldehyde having in. its ortho position a radical. co-ordinatable Withmetaizincombination with carboxylic acid and the amino radical in the; Ot-POSliiOlJ of: the. amino: acid, so thatithe hydrogenatom in the ot-posis tion is dissociated.

The present inventors perceived the structure of the u+position1 of- L-2+pyrrolidone'-5'-carboxylic acid and inferred that the-racemization might not occur in the case of this amino acid since the imino radical of the acid could form no complex compound with the aromatic or heterocyclic aldehyde having in its ortho-position a radi-. cal coordinatable With metal and the metallic ion. The inference was proved positively, establishing that L2- pyrrolidone-S-carboxylic acid is not racemized in the presence of (1) an aldehyde having in its ortho-position a radical co-ordinatable with metal and (2) a metallic ion.

The foregoing is supported by the data in Table 1 obtained from aqueous solutions of L-glutamic acid and L-2-pyrrolidone-5-carboxylic acid each containing salicylaldehyde and a copper ion.

TABLE 1 Racemizaiion ratios (percent) of L-glutamic acid and L- pyrrolr'done-S-darboxylic acid (at C.)

L-glutamic L-pyrrolidonc- Reaction Time (hour) Acid Scarboxylic acid As is obviousfrom Table 1, there is a specific differ-.

encebetween.racemization, through a complex compound formation, of glutamic acid and of pyrrolidone-S-carboxylic acid, the latter not being racemized at all.

Besides, the inventors recognized that the enzymatic activity of Pseudomonas cruciviae is exceptionally powerful. Said activity is hardly affected by the presence of an aromatic or heterocyclic aldehyde or a metallic ion, so that L-glutamic acid is. specifically dehydrated to L-2- pyrrolidone. -5-carboxylic acid. Furthermore, they discovered that by the combination of the racemization reaction through said complex compound formation and the enzymatic conversion reaction of L-glutamic acid into L-2-pyrrolidone-5-carboxylic acid, racemic glutamic acid could be completely converted into L-2-pyrrolidone-5- carboxylic acid. By a concurrent advance of the. both reactions, the reactionequilibrium moves interdependently so. that racemic glutamic acid rapidly changes to L-2- pyrrolidOne-S-carboxylic acid. L-glutamic acid can be thereby easily obtained in higher yields by hydrolyzing the formed L-2-pyrrolidone-S-carboxylic acid to L-glutamic acid. An extremely advantageous novel method for producing L-glutainic acid from racemic glutamic acid has been thus originated.

For the production of L-glutamic acid from racemic glutamic acid inaccordance with the invention, the first step is to act an enzyme material, obtained by cultivation of Pseudom-onas crucz'viae, upon an aqueos solution of racemicglutamic acid in-the presence of (1) an aromatic or heterocyclic aldehyde having inits'ortho-posis tion: a radical coordinatable with metal and (2) a metallic ion; Thereby, L-glutamic acid in the solution is dehydratedby theenzymatic action to L-Z-pyrro-lidbne-S- carboxylic acid; concurrently, the remainingD-glutamic acid is racemized, by movement of the reaction equilibrium, to give racemic glutamic acid. The resulting L-glutamic acid in the racemized' acid is dehydrated as soon as; it forms to yield L-Z-pyrrolidone-S-carboxylic acid; Therefore, the velocity of the racemizationreaction is extraordinarily increased. Furthermore, the de-. hydration: reaction of the L-glutamic acid is extremely rapid since the amounttof L-glutamic acidin' relation to that of enzyme is alwaysso small that the enzyme acts effectively. Thus, racemic glutamic acid is completely converted into Le2 pyrrolidone-5-carboxylic acid in a shorter period. ofitime: bythe. mutual. actionof: ('a). the;

enzymatic conversion of L-glutamic acid into L-Z-pyrrolidone-S-carboxylic acid and (b) the racemization of D-glutamic acid.

In practicing the first step of the invention, it is preferable to add an aromatic or heterocyclic aldehyde and a metallic ion to an aqueous solution containing racemic glutamic acid and then to add an enzyme material to the mixture. Alternatively, the enzyme material may be added to the aqueous solution containing racemic glutamic acid, and then, immediately or after a while, the aromatic or heterocyclic aldehyde and the metallic ion may be added.

The enzyme material employed in the invention may be one which is obtained by cultivation of Pseudomonas cruciviae. The term, enzyme material, used herein involves in its meaning a proliferated culture medium, cells recovered from the culture medium, the cell homogenates obtained by supersonic vibrations, the enzyme obtained therefrom and the like. Pseudomonas cruciviae is a known species, cultures of which are available in public culture collections. It may be isolated from soil and is identified by published descriptions, such as Bergeys Manual of Determinative Bacteriology, Breed, Robert S., Murray, E. G. D., and Smith, Nathan R., seventh edition, The William & Wilkins Company, Baltimore, Md., 1957, for use in the present process.

The aromatic or heterocyclic aldehyde having in its ortho-position a radical coordinatable with metal is exemplified by salicylaldehyde. The term, a radical coordinatable with metal, means a metallizable radical and encompasses the hydroxy group, i.e. OH; alkoxy group, e.g. OCH amino group, e.g. NH N(CH etc. Other examples of the aldehyde are pyridoxal, deoxypyridoxal, pyridoxal phosphate, 2 formyl-3-hydroxy-pyridine, 4-nitrosalicylaldehyde, 6-nitrosalicylaldehyde, Z-aminobenzaldehyde etc. The aromatic or heterocyclic aldehyde may be employed in a concentration of to mol per 1 mol of racemic glutamic acid.

The metallic ion to be added with the aldehyde is one which can be coordinated with the aldehyde. The preferred ions are Ga+++, Cu++, Al+++, Fe++, Fe+++, Zn++, In+++, Ni++, and Co++. The metallic ion may be added in a concentration of to ,4 mol per 1 mol of racemic glutarnic acid. Usually it is added in the form of a salt. Examples of preferred salts are chlorides, e.g. CuCl bromides, e.g. ZnBr iodides, e.g. NiI sulfates, e.g. Al (SO -18H O, Ga (SO nitrates, e.g. ID(NO3)3'3H20; acetates, e.g. CO(C2H302)2'4H20; and double salts, e.g. (NH )Fe(SO -12H O,

Any other salt which contains an above specified metallic ion and which is soluble in water may also be used in the present process.

The reaction conditions of the first step may be varied broadly without inhibiting the enzymatic action. The preferable reaction temperature is from 50 to 70 C. The pH of the reaction mixture at the beginning is preferably about 8. The preferable concentration of the racemic glutamic acid in the reaction mixture is from about 10 to 100 milligrams per milliliter (mg/ml). Usually a reaction period ranging from 4 to 10 hours sufiicies to obtain a quantitative yield.

The second step of the invention is the hydrolysis of the resulting L-Z-pyrrolidone-S-carboxylic acid to L- glutamic acid. This step may be carried out after the isolation of the resulting L-Z-pyrrolidone-S-carboxylic acid from the mixture of the first step, although the latter may be directly hydrolyzed.

The procedure of the isolation of L-2-pyrrolidone-5- carboxylic acid, it it is desired, can be carried out, for example, by extracting the aromatic or heterocyclic aldehyde at pH of about 1.0 with a suitable solvent, such as ether, removing the metallic ion and unreacted amino acid, if any, by means of, e.g., ion-exchange resin and 4 then recovering the L-Z-pyrrolidone-S-carboxylic acid in the solution.

The hydrolysis of L-2-pyrrolidone-5-carboxylic acid in accordance with the second step of the invention is conventional and may therefore be conducted by any of the procedures known to those skilled in the art.

The invention is illustrated by the following examples, which are not to be construed as limitative.

EXAMPLE 1 A culture medium containing 5 g. (grams) of meat extract, 2 g. of yeast extract, 1 g. of K HPO 0.25 g. of MgSO -7H O, 10 g. of NZ-amine (trade name for an enzymatic digest of casein for microbiological use), 10 g. of glucose, and water to make 1 liter, having a pH of 7.2, is inoculated with Pseudomonas cruciviae and cultivated at a temperature of 38 C. for 24 hours under aerobic conditions. After completion of the cultivation, the cells are separated from the culture medium by centrifugation at 5 C., washed with saline water, and dried by freezedrying. The dried cells are thus obtained.

To 2000 ml. of an aqueous solution containing 100 g. of racem-ic glutamic acid are added 10 g. of 4-nitrosalicylaldehyde and 5 g. of cupric chloride. After adjusting the pH to 8.0, 1 g. of dried cells obtained above is added thereto. The reaction is allowed to proceed at a temperature of 60 C. for 5 hours, whereby glutarnic acid in the solution is substantially completely converted into L-2-pyrrolidone-5-carboxylic acid. After termination of the reaction, the solution at a pH of 1.0 is extracted with half its volume of ether (diethyl ether) to recover 4-nitrosalicylaldehyde. The residue of the extraction is passed through a column filled with 100 g. of a strongly acidic ion exchange resin, Amberlite IR-120 (trade name for sulfonated polystyrene type resin), which is previously regenerated with 10% (by Weight) hydrochloric acid and washed with water, whereby cupric ion and unreacted D- glutamic acid are wholly removed by adsorption. The effluent (2500 ml.) is concentrated to 400 ml. under reduced pressure and, at the same time, hydrolyzed by addition of 100 ml. of 32% (by weight) hydrochloric acid. When adjusting the pH to 3.2 with sodium hydroxide, crystals isolate. The crystals are separated and dried to yield 90 g. of L-glutamic acid.

EXAMPLE 2 From cells prepared in the same way as those of Example 1, a crude enzyme preparation is prepared as follows: (.i) S-gram portions of the lyophilized cells are treated with supersonic vibrations (10 kilo-cycles per second) at 0 to 3 C. in milliliters of M/l5 phosphate buffer for 30 minutes. (ii) After centrifugation at 8000 gravitational force for 10 minutes, the supernatant is allowed to stand in a water bath at 70 C. for 30 minutes. (iii) After centrifugation the supernatant milliliters) is treated with 45 milliliters of saturated ammonium sulfate solution at 0 C. (to give 0.28 saturation). After 20 hours, the precipitate is removed and discarded. The resulting supernatant (127 milliliters) is then brought to 0.62 saturation by addition of ammonium sulfate powder. The resulting precipitate is removed by centrifugation and lyophilized.

To 1000 ml. of an aqueous solution containing 56 g. of racemic glutamic acid are added 5 g. of 6-nitrosalicylaldehyde and 15 g. of alum [AlK(SO -12H O]. After addition of 200 mg. of crude enzyme preparation of Pseudomonas cruciviae obtained as above, the reaction is allowed to proceed at a temperature of 50 C. and at a pH of 7.5 for 10 hours, whereby glutamic acid in the solution is converted into L-Z-pyrrolidone-5-carboxylic acid. The reaction mixture is treated in the same way as that of Example 1. L-glutamic acid (48.5 g.) is obtained. This corresponds to 86.5% of the theoretical yield.

EXAMPLE 3 To 1500 ml. of an aqueous solution containing 75 g.

sponsor;

ofiracemic: glutarnic acid; areadded 7g. of salicylaldehyde; 3v g. of cupricchloride and' 1 g; of dried cells of Rseudamonasccruciviae obtained as in Example 1. reaction is allowed toproceed at a temperature of 70 C. and at a pH of 7.7 for12 hours. The reaction mixture is treated in the same way as that ofExample 1. L-glutamic acid (69 g.) is obtained. This corresponds to 92% of the theoretical yield.

It is thought that the invention and its advantages will be understood from the foregoing description, and it is apparent that various changes may be made in the process and composition without departing from the spirit and scope of the invention or sacrificing its material advantages, the process and composition hereinbefore described being merely illustrative of preferred embodiments of the invention.

What is claimed is:

1. A method of producing L-glutamic acid from racemic glutarnic acid which comprises: (1) preparing a reaction mixture by combining with an aqueous solution of racemic glutarnic acid (a) aldehyde having in a position ortho to the aldehyde group a radical coordinatable with metal, said aldehyde being a member selected from the group consisting of aromatic and heterocyclic aldehydes and (b) metallic ion coordinatable with the aldehyde; (2) subjecting said reaction mixture to the action of enzyme of Pseudomonas cruciviae, whereby the racemic glutarnic acid is converted into L-2pyrrolidone-5- carboxylic acid; and (3) hydrolyzing the L-2-pyrrolidone-S-canboxylic acid to L-glutam-ic acid.

2. The method according to claim 1 wherein the aldehyde is a member selected from the group consisting of salicylaldehyde, pyridoxal, S-deoxypyridoxal, pyridoxal phosphate, 2-formyl-3-hydroxypyridine, 4-nitrosalicyladehyde, 6-nitrosalicylaldehyde and Z-aminobenzaldehyde.

3. The method according to claim 1 wherein the concentration in the reaction mixture of the aldehyde is from to mol per 1 mol of racemic glutamic acid.

4. The method according to claim 1 wherein the metallic ion is a member selected from the group consisting of cupric, gallic, aluminum, ferrous, \ferric, zinc, indium, nickelous, and cobaltous ions.

5. The method according to claim 1 wherein the concentration in the reaction mixture of metallic ion is from ,5 to 4 mol per 1 mol of racemic glutamic acid.

6. The method according to claim 1 wherein the enzyme is in the form of dried cells.

7. A method of producing L-glutamic acid from racemic glutamic acid which comprises: (1) preparing a reaction mixture by combining with an aqueous solution of racemic glutarnic acid (a) aromatic aldehyde having in its ortho-position a metallizable radical and (b) metallic ion coordinatable with the aldehyde; (2) subjecting said reaction mixture to the action of enzyme of Pseudomonas cruciviae, whereby the racemic glutamic acid is converted into L-Z-pyrrolidone-S-carboxylic acid; and (3) hydrolyzing the L-Z-pyrrolidone-Scanboxyiic acid to L-glutamic acid.

8. A method of producing L-glutamic acid from racemic glutamic acid which comprises: (1) preparing a reaction mixture by combining with an aqueous solution of racemic glutamic acid (a) heterocyclic aldehyde having in its ortho-position a metallizable radical and (b) metallic ion coordinatable with the aldehyde; (2) subjecting said reaction mixture to the action 0t enzyme of Pseudomonas cruciviae, whereby the racemic glutarnic acid is converted into L-2-pyrrolidone-S-carboxylic acid; and 3) hydrolyzing the L-2-pyrrolidone-S-canboxylic acid to L-glutamic acid.

9. A method of producing L-glutamic acid from racemic glutarnic acid which comprises: (1) preparing a reaction mixture by combining with an aqueous solution of racemic glutarnic acid (a) salicyialdehyde and (b) metallic ion coordinatable with the salicylaldehyde; (2) subjecting said reaction mixture to the action of enzyme of'Pseudomonas crucl'viae, whereby the racemic: glutarnic acid is converted into L-Z-pyrrolidone-S-carboxylic acid; and (3) hydrolyzing'the-L 2 py-rrolidone-S-carboxylic acid to L-glutamic acid;

10. A method of producing L-glutamic acid from racemic. glutarnic acid which comprises: (1) preparing a reaction mixture by combining with an aqueous solution of racemic glutamic acid (a) pyridoxal and (b) metallic ion coordinatable with the pyridoxal; (2) subjecting said reaction mixture to the action of enzyme of PseudomOn-as crucrlviae, whereby the racemic glutarnic acid is converted into L-Z-pyrrolidone-S-carboxylic acid; and (3) hydrolyzing the L-2-pyrrolidone-5-carboxylic acid to L-glutamic acid.

11. A method of producing L-glutamic acid from racemic glutamic acid which comprises: (1) preparing a reaction mixture 'by combining with an aqueous solution of racemic glutamic acid (a) S-deoxypyridoxal and (b) metallic ion coordinatable with the S-deoxypyridoxal; (2) subejcting asid reaction mixture to the action of enzyme of Pseudomonas cruciviae, whereby the racemic glutarnic acid is converted into L-2-pyrrolidone-5-carboxylic acid; and (3) hydrolyzing the L-2-pyrrolidone-5-carboxylic acid to L-glutamic acid.

12. A method of producing L-glutamic acid from race mic glutarnic acid which comprises: 1) preparing a reaction mixture by combining with an aqueous solution of racemic glutamic acid (a) pyridoxal phosphate and (b) metallic ion coordinatable with the pyridoxal phosphate; (2) subjecting said reaction mixture to the action of enzyme of Pscudomonas cruciviae, whereby the racemic glutamic acid is converted into L-Z-pyrrolidone-S-carboxylic acid; and (3) hydrolyzing the L-2-pyrrolidone-5- carboxylic acid to L-glutamic acid.

13. A method of producing L-glutamic acid from racemic glutamic acid which comprises: (1) preparing a reaction mixture by combining with an aqueous solution of racemic glutarnic acid (a) 2-formyl-3-hydroxypyridine and (b) metallic ion coordinatable with the 2-formyl-3 hydroxypyn'dine; (2) subjecting said reaction mixture to the action of enzyme of Pseudomonas cruciviae, whereby the racemic glutamic acid is converted into L-Z-pyrrolidone-S-carboxylic acid; and (3) hydrolyzing the L2- pyrrolidone-S-carboxylic acid to L-glutamic acid.

14. A method of producing L-glutamic acid from racemic acid which comprises: (1) preparing a reaction mixture by combining with an aqueous solution of racemic glutamic acid (a) 4-nitrosalicylaldehyde and (b) metallic ion coordinatable with the 4-nitrosal-icylaldehyde; (2) subjecting said reaction mixture to the action of enzyme of Pseudomonas cruciviae, whereby the racemic glutamic acid is converted into L2-pyrrolidone-5-carboxylic acid; and (3) hydrolyzing the L-2-pyrrolidone-5-carboxylic acid to L-glutamic acid.

15 .A method of producing L-glutamic acid from racemic glutamic acid which com-prises: (l) preparing a reaction mixture by combining with an aqueous solution of racemic glutarnic acid (a) 6-nitrosalicylaldehyde and (b) metallic ion coordinatable with the G-nitrosalicylaldehyde; (2) subjecting said reaction mixture to the action of enzyme of Pseudomonas cruciviae, whereby the racemic glutamic acid is converted into L-Z-pyrrolidone-S-carboxylic acid; and (3) hydrolyzing the L-Z-pyrrolidone-S- carboxylic acid to L-glutamic acid.

16. A method of producing L-glutamic acid from racemic glutamic acid which comprises: (1) preparing a reaction mixture by combining with an aqueous solution of racemic glutamic acid (a) Z-aminobenzaldehyde and (b) metallic ion coordinatable with the Z-aminobeuzaldehyde; (2) subjecting said reaction mixture to the action of enzyme of Pseudomonas aruciviae, whereby the racemic glutarnic acid is converted into L-Z-pyrrolidone-S-carboxylic acid; and (3) hydrolyzing the L-2-pyrrolidone-5- carboxylic acid to L glutamic acid.

17. A method of producing L-2-pyrrolidone-5-carboxylic acid which comprises: 1) preparing a reaction mixture by combining with an aqueous solution of racemic glutamic acid (a) aldehyde having in a position ortho to the aldehyde group a radical coordinatable with metal, said aldehyde being a member selected from the group consisting of aromatic and heterocyclic aldehydes, and

8 (b) metallic ion coordinatable with the aldehyde and (2) subjecting said reaction mixture to the action of enzyme of Pseudomonas cruciviae, whereby the racemic glutamic acid is converted into L-2-pyrrolidone-5-carboxylic acid.

No references cited. 

1. A METHOD OF PRODUCING L-GLUTAMIC ACID FROM RACEMIC GLUTAMIC ACID WHICH COMPRISES: (1) PREPARING A REACTION MIXTURE BY COMBINING WITH AN AQUEOUS SOLUTION OF RACEMIC GLUTAMIC ACID (A) ALDEHYDE HAVING IN A POSITION ORTHO TO THE ALDEHYDE GROUP A RADICAL COORDINATABLE WITH METAL, SAID ALDEHYDE BEING A MEMBER SELECTED FROM THE GROUP CONSISTING OF AROMATIC AND HETEROCYCLIC ALDEHYDES AND (B) METALLIC ION COORDINATABLE WITH THE ALDEHYDE, (2) SUBJECTING SAID REACTION MIXTURE TO THE ACTION OF ENZYME OF PSEUDOMONAS CRUCIVIAE, WHEREBY THE RACEMIC GLUTAMIC ACID IS CONVERTED INTO L-2-PYRROLIDONE-5CARBOXYLIC ACID, AND (3) HYDROLYZING THE L-2-PYRROLIDONE-5-CARBOXYLIC ACID TO L-GLUTAMIC ACID 